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• W1645330008 abstract "Non-Technical Summary In multiple brain regions, endogenous cannabinoids suppress inhibitory synaptic transmission; however, the biochemical/molecular pathways for endocannabinoid synthesis are poorly understood. Endocannabinoid signalling may be crucial for microcircuit function in the prefrontal cortex (PFC), a cortical region involved in complex behaviours. However, endocannabinoid signalling remains largely unexplored in the PFC. Using enzymatic inhibitors, we show that modulation of inhibitory synaptic transmission in PFC neurons is mediated by the endocannabinoid 2-arachidonoylglycerol synthesized postsynaptically. Interestingly, diacylglycerol lipase (DAGL), the 2-arachidonoylglycerol synthesis enzyme, has two isoforms: DAGLα and DAGLβ. Studying PFC neurons from DAGLα−/−, DAGLβ−/− and wild-type mice, we show that only DAGLα is involved in the suppression of inhibitory transmission in the PFC. Abstract Depolarization-induced suppression of inhibition (DSI) is a prevailing form of endocannabinoid signalling. However, several discrepancies have arisen regarding the roles played by the two major brain endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide, in mediating DSI. Here we studied endocannabinoid signalling in the prefrontal cortex (PFC), where several components of the endocannabinoid system have been identified, but endocannabinoid signalling remains largely unexplored. In voltage clamp recordings from mouse PFC pyramidal neurons, depolarizing steps significantly suppressed IPSCs induced by application of the cholinergic agonist carbachol. DSI in PFC neurons was abolished by extra- or intracellular application of tetrahydrolipstatin (THL), an inhibitor of the 2-AG synthesis enzyme diacylglycerol lipase (DAGL). Moreover, DSI was enhanced by inhibiting 2-AG degradation, but was unaffected by inhibiting anandamide degradation. THL, however, may affect other enzymes of lipid metabolism and does not selectively target the α (DAGLα) or β (DAGLβ) isoforms of DAGL. Therefore, we studied DSI in the PFC of DAGLα−/− and DAGLβ−/− mice generated via insertional mutagenesis by gene-trapping with retroviral vectors. Gene trapping strongly reduced DAGLα or DAGLβ mRNA levels in a locus-specific manner. In DAGLα−/− mice cortical levels of 2-AG were significantly decreased and DSI was completely abolished, whereas DAGLβ deficiency did not alter cortical 2-AG levels or DSI. Importantly, cortical levels of anandamide were not significantly affected in DAGLα−/− or DAGLβ−/− mice. The chronic decrease of 2-AG levels in DAGLα−/− mice did not globally alter inhibitory transmission or the response of cannabinoid-sensitive synapses to cannabinoid receptor stimulation, although it altered some intrinsic membrane properties. Finally, we found that repetitive action potential firing of PFC pyramidal neurons suppressed synaptic inhibition in a DAGLα-dependent manner. These results show that DSI is a prominent form of endocannabinoid signalling in PFC circuits. Moreover, the close agreement between our pharmacological and genetic studies indicates that 2-AG synthesized by postsynaptic DAGLα mediates DSI in PFC neurons. The two major endocannabinoids found in brain, 2-arachidonoylglycerol (2-AG) and anandamide, are effective agonists of the primary brain cannabinoid receptor, the cannabinoid receptor 1 (CB1R) (Kano et al. 2009). Endocannabinoids are released rapidly via non-vesicular mechanisms following stimulation of their synthesis, and retrogradely inhibit neurotransmitter release via presynaptic CB1Rs (Wilson & Nicoll, 2001). Among other stimuli, endocannabinoid synthesis is activated by postsynaptic depolarization, which produces a CB1R-dependent retrograde suppression of GABA release. Endocannabinoid-mediated depolarization-induced suppression of inhibition (DSI) is synapse-specific and short-lasting, decaying within seconds (Katona et al. 1999; Nyiri et al. 2005; Glickfeld & Scanziani, 2006; Galarreta et al. 2008). In the hippocampus, cerebellum and striatum, multiple properties of DSI were previously studied, including the contribution of 2-AG and anandamide. Some studies using endocannabinoid synthesis inhibitors suggested that DSI requires 2-AG without a significant anandamide contribution (Kano et al. 2009). However, several experimental discrepancies have arisen (Di Marzo, 2011). For example, in some studies, 2-AG synthesis inhibitors failed to affect DSI even though they blocked other forms of endocannabinoid-mediated synaptic modulation (Chevaleyre & Castillo, 2003; Safo & Regehr, 2005; Min et al. 2010b). Such discrepancies may be due to the use of different enzymatic inhibitors, some of which could have non-specific effects, the use of cell cultures versus acute slices, or regional differences in the roles of 2-AG versus anandamide in DSI. In the prefrontal cortex (PFC), a neocortical region with substantially different circuitry to hippocampus or cerebellum, endocannabinoid-mediated signalling remains largely unexplored, although the PFC contains molecular components of the endocannabinoid system, including CB1Rs (Eggan & Lewis, 2007; Lafourcade et al. 2007; Burston et al. 2010; Chiu et al. 2010), fatty acid amide hydrolase and monoacylglycerol lipase, the anandamide- and 2-AG-degrading enzymes, respectively, and diacylglycerol lipase (DAGL), the key enzyme for 2-AG synthesis (Hansson et al. 2007; Lafourcade et al. 2007; Volk et al. 2010). Genes for two DAGL isoforms with very similar enzymatic activity, DAGLα and DAGLβ, have been cloned (Bisogno et al. 2003). Interestingly, in hippocampal (Katona et al. 2006; Yoshida et al. 2006; Ludanyi et al. 2011) and prefrontal (Lafourcade et al. 2007) pyramidal cells, DAGLα is highly expressed in dendritic spines, where it can retrogradely modulate glutamate release (Katona & Freund, 2008). However, DAGLα in dendritic spines is ultrastructurally distant from most GABA synapses and is thus unlikely to contribute to DSI, since the lipid-soluble nature of 2-AG severely limits its diffusion in the extracellular space. Notably, DAGLα was reported to be undetectable at CB1R-containing GABA synapses in PFC (Lafourcade et al. 2007), suggesting that 2-AG synthesized by DAGLα mostly or exclusively modulates glutamate synapses. In fact, in PFC endocannabinoids modulate excitatory synaptic transmission (Lafourcade et al. 2007, 2011), but whether endocannabinoids produce DSI in PFC circuits has not been reported. The very low levels of DAGLα near CB1R-containing GABA synapses in PFC (Lafourcade et al. 2007) suggest that DSI in PFC might depend on DAGLβ, although the ultrastructural localization of DAGLβ has not been determined. Alternatively, DSI in PFC may depend on anandamide instead of 2-AG. Anandamide mediates some forms of suppression of inhibition onto pyramidal neurons (Lourenço et al. 2011) and, interestingly, plays a critical role in PFC circuit function. For example, application of exogenous anandamide to the PFC or manipulating the effects of endogenous anandamide in the PFC, both produce significant behavioural effects (Rubino et al. 2008; Aguiar et al. 2009; Lisboa et al. 2010). Two recent studies showed that DSI is abolished in hippocampus and cerebellum of DAGLα-deficient mice, suggesting that DSI requires 2-AG synthesized via DAGLα (Gao et al. 2010; Tanimura et al. 2010). However, in such studies DAGLα deficiency decreased both 2-AG and anandamide levels in total brain tissue (Gao et al. 2010) or specifically in hippocampus and cerebellum (Tanimura et al. 2010). Thus, such studies could not definitively exclude a contribution of anandamide to DSI (Min et al. 2010a; Di Marzo, 2011). Moreover, those studies did not examine endocannabinoid signalling in neocortical circuits. To study endocannabinoid-mediated modulation of inhibitory synaptic transmission in PFC, we used electrophysiology, pharmacology and genetically modified mice. We show that in PFC pyramidal neurons DSI occurs independently of anandamide signalling or DAGLβ activity, but requires 2-AG synthesized by DAGLα localized in postsynaptic pyramidal cells. Brain slices were prepared from the frontal cortex of mice deeply anaesthetized with isoflurane and decapitated following procedures in accordance with NIH guidelines and approved by the University of Pittsburgh's Institutional Animal Care and Use Committee. The authors have read, and the experiments comply with the policies and regulations of The Journal of Physiology, given by Drummond (2009). In most experiments we used male C57BL6 mice (Charles River), 1–4 months of age. In the experiments conducted with tissue from DAGL-deficient mice and their wild-type littermates, we used 5- to 6-week-old male mice. The brain was quickly removed and immersed in ice-cold, choline-based artificial cerebrospinal fluid (ACSF) containing (mm): 110 choline chloride, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 1.25 NaH2PO4, 11.6 sodium ascorbate, 3.1 sodium pyruvate and 25 d-glucose, pH 7.3–7.4, and continuously bubbled with 95% O2 and 5% CO2. The frontal cortex was sectioned into 300 μm-thick slices in the coronal plane, using a vibrating microtome (VT1000S, Leica Microsystems). Slices were immediately incubated for 30–60 min in a chamber maintained at 36°C and filled with standard ACSF (mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 25 NaHCO3, 1.25 Na2HPO4 and 10 glucose, pH 7.3–7.4, and continuously bubbled with 95% O2 and 5% CO2. Following incubation, brain slices were stabilized at room temperature in the same solution for at least 50 min. Slices were then transferred to an electrophysiology recording chamber where the submerged slices were superfused at a flow rate of 3–4 ml min−1 (to record carbachol-induced spontaneous (s)IPSCs or sIPSPs, see Hájos & Mody, 2009) or 2 ml min−1 (to record agatoxin-resistant evoked IPSCs) with ACSF gassed with 95% O2 and 5% CO2 at 30–32°C. AMPA receptor-mediated transmission was blocked routinely by adding 10 μm 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX). CNQX, carbachol, tetrahydrolipstatin (THL), N-arachidonoyl maleimide (NAM), WIN55212-2, and AM251 were purchased from Sigma Chemical Company, St Louis, MO, USA or Tocris Bioscience, Ellisville, MO, USA. SR141716A was provided by Bristol-Myers Squibb. Biocytin was purchased from Sigma or Invitrogen, Carlsbad, CA, USA. ω-Conotoxin-GVIA and ω-agatoxin-IVA were from Bachem, Torrance, CA, USA. URB597 was from Cayman Chemicals, Ann Arbor, MI, USA. Patch-pipette, tight-seal whole-cell recordings were obtained from pyramidal cells in layers 2–3 of the infralimbic (IL), prelimbic (PL) or anterior cingulate (AC) regions of the mouse medial frontal cortex, here collectively referred to as mouse PFC (Fig. 1A). Cells were visualized using Olympus or Zeiss microscopes equipped with infrared illumination and differential interference contrast videomicroscopy. IPSCs were recorded using pipettes pulled from borosilicate glass having a resistance of 2–5 MΩ when filled with the following solution (mm): 120 CsCl, 2 KCl, 10 Hepes, 0.2 EGTA, 4 MgATP, 0.3 NaGTP and 0.5% biocytin, pH adjusted to 7.2–7.4 using CsOH. IPSPs were recorded using pipettes filled with (mm): 120 KCl, 10 NaCl, 10 Hepes, 0.2 EGTA, 4 MgATP, 0.3 NaGTP, 14 phosphocreatine and 0.5% biocytin. The intrinsic excitability of PFC pyramidal cells (Fig. 9) was determined using pipettes filled with a solution containing (mm): 120 potassium gluconate, 10 NaCl, 10 Hepes, 0.2 EGTA, 4.5 MgATP, 0.3 NaGTP and 14 sodium phosphocreatine. Recordings were obtained using Multiclamp 700A or 700B amplifiers (Axon Instruments). Signals were low-pass filtered at 4 kHz and digitized at 10 kHz using Power 1401 data acquisition interfaces (Cambridge Electronic Design, Cambridge, UK). Data acquisition and analysis were performed using Signal 4 software (Cambridge Electronic Design). Depolarizing steps do not suppress sIPSCs recorded from layer 2/3 PFC neurons in the absence of carbachol A, diagram showing the location of recorded pyramidal neurons in layers 2/3 of the mouse medial PFC. AC: dorsal anterior cingulate cortex, PL: prelimbic cortex, IL: infralimbic cortex, CC: corpus callosum. B, example traces of sIPSCs recorded from layer 2/3 PFC pyramidal neurons. Note that GABA synapses elicited inward IPSCs due to the high chloride concentrations in the pipette recording solution. C, effect of depolarizing steps (–80 to 0 mV for 4 s) on sIPSCs recorded from layer 2/3 pyramidal neurons. The depolarizing steps did not alter the sIPSC frequency or amplitude, suggesting that the sIPSCs were mostly produced by cannabinoid-insensitive synapses. Here and in other figures, the current elicited by the depolarizing steps has been blanked. D, quantification of the effect of depolarizing steps on sIPSCs recorded from PFC neurons. As described in Methods, for each neuron the baseline sIPSCs (sIPSCs recorded prior to the depolarizing steps) were averaged and all IPSCs (baseline and post-depolarization) were normalized relative to the average baseline value. Here and in all other figures, a normalized sIPSC value of 1 means no change in sIPSC amplitude; a value of 0 means 100% sIPSC suppression. DAGLα deficiency reduced the excitability of PFC pyramidal cells A, example traces illustrating the membrane potential response of wild-type and DAGLα−/− PFC neurons to injection of depolarizing input current steps of increasing amplitude (50, 150 and 250 pA). Note the lower excitability of the PFC neurons from DAGLα−/− mice revealed as smaller number of spikes for identical level of input current. To test the effects of input current and genotype on the excitability of PFC pyramidal neurons, we recorded from 7 layer 2/3 PFC neurons from wild-type mice and 8 neurons from DAGLα−/− littermates. Input current was increased in 10 pA increments up to 400 pA above threshold. To monitor recording conditions over time, a small (–50 pA) hyperpolarizing current step was injected prior to the depolarizing stimulation. Note the weaker response to the hyperpolarizing step in neurons from DAGLα−/− mice. Differences in the excitability of wild-type and DAGLα−/− neurons were examined using current clamp recordings from n= 7 wild-type and n= 8 DAGLα−/− neurons. B, firing frequency (prior to onset of spike-frequency adaptation) plotted as a function of input current. Note that PFC pyramidal neurons from DAGLα−/− mice fired at lower frequency throughout a wide range of input current. Here and in panels C, D and E, input current is normalized relative to each neuron's current threshold, (the minimum level of current necessary to fire action potentials). Two-factor ANOVA showed significant effects of input current (F(40,533)= 31.032, P < 0.00001) and genotype (F(1,533)= 207.5, P < 0.000001) on the firing frequency, as well as a significant current–genotype interaction (F(48,629)= 1.9234, P < 0.0005). C, voltage–current plot illustrating the membrane potential response of wild-type and DAGLα−/− neurons to injection of hyperpolarizing current steps. Wild-type neurons displayed a steeper relationship, suggesting that DAGLα−/− neurons had lower membrane resistance. Two-factor ANOVA indicated a significant effect of genotype on the membrane potential response to current injection (input current: F(4,65)= 43.5, P < 0.0001; genotype: F(1,65)= 4.9353, P= 0.0298). D, PFC neurons from DAGLα-deficient mice had higher voltage threshold for firing an action potential (first action potential fired in response to a current step) throughout a wide range of input currents. Two-factor ANOVA indicated a significant effect of genotype on firing threshold (F(1,403)= 170.14, P < 0.0001). E, for input currents up to ∼100 pA above current threshold, DAGLα−/− PFC neurons showed stronger spike-frequency adaptation, as revealed by a lower adaptation ratio, measured as the ratio between the first and last inter-spike interval (an adaptation ratio value of 1 means no adaptation and values below 1 mean a stronger spike-frequency adaptation). Two-factor ANOVA showed significant effects of input current (F(48,629)= 14.115, P < 0.00001) and genotype (F(1,629)= 48.566, P < 0.00001) on the adaptation ratio, as well as a significant current–genotype interaction (F(48,629)= 1.9234, P < 0.0005). Voltage clamp The pipette capacitance was compensated and series resistance was continuously monitored but was not compensated. Only recordings with a stable series resistance of less than 20 MΩ were used for analysis. To record IPSCs, pyramidal cells were held at –80 mV. Current clamp Series resistance and pipette capacitance were monitored and cancelled using bridge and capacitance neutralization. Membrane potential was not corrected for liquid junction potential. The membrane potential was maintained near –75 mV with current injection. Extracellular stimulation Agatoxin-resistant IPSCs were evoked by focal stimulation using electrodes fabricated with theta-type capillary glass pulled to an open tip diameter of ∼3–5 μm and filled with oxygenated ACSF. Silver wires inserted into the theta glass were connected to a stimulus isolation unit (World Precision Instruments) commanded by TTL pulses. Stimulation at 0.2 Hz (duration = 100 μs, amplitude = 10–100 μA) was delivered after placing the stimulation pipette near the soma of the recorded pyramidal cell. Biocytin-filled neurons were visualized using the Vectastain Elite ABC kit (Vector Laboratories) and their axonal and dendritic trees reconstructed using the Neurolucida Tracing System (Microbrightfield Bioscience) as described previously (Gonzalez-Burgos et al. 2009). In the majority of the experiments, we studied DSI of GABAA receptor-mediated IPSCs induced in pyramidal cells by application of the cholinergic agonist carbachol. Previous studies showed that carbachol-induced IPSCs display strong DSI, consistent with the idea that carbachol activates cholecystokinin-containing interneurons which furnish cannabinoid-sensitive GABA synapses (however, see Gulyas et al. 2010; Szabóet al. 2010). We used 20 μm carbachol, a concentration that produces stable increases in sIPSC amplitude and frequency in submerged slices (Hájos & Mody, 2009). In PFC pyramidal neurons, carbachol application rapidly increased the IPSC frequency in a manner that was sensitive to blockade of N-type voltage-dependent Ca2+ channels (Fig. 2), which control GABA release from cannabinoid-sensitive synapses (Freund & Katona, 2007). In preliminary experiments we determined that, as reported previously (Hájos & Mody, 2009), the amplitude and frequency of carbachol-induced sIPSCs reached steady-state values within 1–5 min after the start of carbachol application (see Fig. 2B). Therefore, the depolarizing commands (–80 to 0 mV, 4 s) were applied to induce DSI starting at 5 min from the beginning of carbachol application. The effects of depolarizing steps on carbachol-induced IPSCs were tested repeating the depolarizing step protocol 3–6 times in each pyramidal neuron. Consistent with recent findings suggesting that some principal neurons receive cholecystokinin-positive DSI-sensitive inputs whereas others do not (Varga et al. 2010), in some layer 2/3 PFC pyramidal neurons DSI of carbachol-induced (evoked) IPSCs (eIPSCs) was absent. The results obtained from cells that were apparently DSI-negative were still included in the data analysis, thus somewhat underestimating the magnitude of IPSC suppression by endocannabinoids in DSI-positive cells. Endocannabinoid-mediated suppression of sIPSCs recorded in the presence of carbachol A, sIPSCs recorded from a PFC pyramidal neuron in control conditions (top panel), after application of 20 μm carbachol (middle panel) and following additional application of the N-type calcium channel blocker ω-conotoxin GVIA (1 μm). Note that ω-conotoxin GVIA application reversed the increase in sIPSC amplitude and frequency produced by carbachol. B, a plot of sIPSC amplitude versus time, showing the increase in sIPSC amplitude by carbachol for the neuron in A. Following the increase in sIPSC amplitude and frequency by carbachol, application of a depolarizing step induced DSI. Once sIPSCs recovered from DSI, ω-conotoxin GVIA was applied, which reversed the effects of carbachol on sIPSC amplitude. C, bar graph summarizing the effects of carbachol application on sIPSC frequency (P < 0.05, Student's t test). D, depolarizing steps (–80 to 0 mV, 4 s) produced a transient suppression of carbachol-induced sIPSCs. Here and in E, the insets show baseline sIPSCs and post-depolarization sIPSCs on an expanded time scale. E, the CB1R antagonist SR141716A (10 μm) abolished suppression of carbachol-induced sIPSC by depolarizing steps. F, left panel: plot of normalized sIPSC amplitude versus time showing the time course of sIPSC suppression by depolarizing steps, in control conditions (vehicle DMSO 0.1%) and in the presence of the CB1R antagonist SR141716A (10 μm); right panel: bar graph summarizing the sIPSC suppression in control conditions versus CB1R blockade with SR141716A (10 μm), P < 0.01 compared with DMSO, Student's t test. In other experiments (Fig. 9), we examined DSI of IPSCs evoked in PFC pyramidal cells by focal extracellular stimulation (eIPSCs) of perisomatic GABA synapses in the presence of ω-agatoxin-IVA (250 nm). This toxin blocks GABA release from the cannabinoid-insensitive GABA synapses in various brain regions, including those of parvalbumin-containing neurons in PFC (Zaitsev et al. 2007), thus producing agatoxin-resistant eIPSCs that display significant DSI (Wilson et al. 2001). Agatoxin-resistant IPSCs were evoked every 5 s. After five stimuli were delivered to obtain a baseline of IPSC amplitude, a depolarizing command (–80 to 0 mV, 4 s) was applied, followed by 20 additional stimuli delivered to determine recovery from DSI. Such a sequence of five baseline eIPSCs, followed by the voltage command and recovery eIPSCs, was repeated at least 6 times for each neuron and eIPSC amplitude measures were averaged for all repetitions for each cell. To study DSI induced by action potential firing (Fig. 10), IPSPs were induced in pyramidal cells by application of carbachol. The experiment design was similar to that employed for carbachol-induced IPSCs, except that the depolarizing command was replaced by trains of short suprathreshold current steps evoked at 20 Hz at different durations. DAGLα deficiency abolishes DSI induced by action potential-mediated stimulation of endocannabinoid synthesis A, top panel: trains of supra-threshold current steps elicited at 20 Hz produced a transient suppression of IPSPs (arrow) induced by application of 20 μm carbachol. Note that together with IPSP suppression, the action potential trains produced a prolonged hyperpolarization consistent with the outward current observed following depolarizing steps in voltage clamp recordings (see Fig. 7). GABA synapses elicited depolarizing IPSPs due to the high chloride concentrations in the pipette recording solution (see Methods). The depolarizing IPSPs occasionally elicited action potentials (marked by *). Spikes were truncated for clarity. Bottom panel: sub-threshold depolarizing current steps delivered in an alternating manner with the supra-threshold steps to the same PFC pyramidal cells of the top panel failed to induce IPSP suppression. In A, C and E action potentials were truncated. B, bar graph summarizing the IPSP suppression produced by alternating supra-threshold versus sub-threshold depolarizing current steps. **P < 0.01 compared with supra-threshold steps, t= 3.788, P < 0.002, paired samples Student's t test, n= 10 cells. C, example of the effect of action potential firing (4 s at 20 Hz) on carbachol-induced IPSPs recorded from a wild-type pyramidal neuron. D, time course plots showing the effects of action potential firing at 20 Hz for different durations on carbachol-induced IPSPs recorded from wild-type neurons. The normalized IPSP values at each time point were compared with the baseline value right before the onset of 20 Hz stimulation. The open symbols indicate significant differences (P < 0.05) compared with last baseline value; post hoc comparisons with Dunnett's test, after single-factor repeated measures ANOVA (F and P values for the ANOVA are shown in the graphs). Note the progressive increase in the duration of statistically significant IPSP suppression with increasing duration of the action potential trains at 20 Hz. E, example of the effect of action potential firing (4 s at 20 Hz) on carbachol-induced IPSPs recorded from a DAGLα−/− pyramidal neuron. F, time course plots showing the effects of action potential firing at 20 Hz for different durations on carbachol-induced IPSPs recorded from DAGLα−/− neurons. Note the absence of significant IPSP suppression compared with the baseline, independent of stimulus duration. Single-factor repeated measures ANOVA indicated no significant IPSP suppression (F and P values for the ANOVA are shown in the graphs. G, graph summarizing the IPSP suppression versus duration of 20 Hz stimulation in wild-type and DAGLα−/− neurons. Two-factor ANOVA indicated a statistically significant effect of genotype on IPSP suppression, F(1,184)= 25.244, P < 0.001. *Significant IPSP suppression compared with baseline IPSP value, P < 0.05 Dunnett's test. #Significantly different compared with IPSP suppression value for the same stimulus duration in wild-type neurons, P < 0.05 Bonferroni or Fisher's LSD post hoc comparison (wild-type neurons, 0.1 s: –0.016 ± 0.042, n= 17; 0.2 s: 0.025 ± 0.045, n= 17; 0.5 s: 0.079 ± 0.044, n= 31; 1.0 s: 0.234 ± 0.060, n= 15; 2.0 s: 0.230 ± 0.044, n= 15; 4.0 s: 0.269 ± 0.064, n= 15; 6.0 s: 0.219 ± 0.069, n= 15; DAGLα−/− neurons: 0.1 s: –0.118 ± 0.042, n= 6; 0.2 s: –0.101 ± 0.069, n= 6; 0.5 s: 0.005 ± 0.039, n= 17; 1.0 s: 0.076 ± 0.024, n= 11; 2.0 s: –0.025 ± 0.040, n= 11; 4.0 s: 0.095 ± 0.042, n= 11; 6.0 s: 0.006 ± 0.074, n= 11). Data were analysed using Signal 4 (Cambridge Electronic Design) and Mini Analysis (Synaptosoft). To assess DSI of sIPSCs induced by carbachol, data were acquired in 120 s-long traces, with application of the 4 s depolarizing step (–80 to 0 mV) to stimulate endocannabinoid synthesis starting at 20 s from the trace onset (see Fig. 1C). For each neuron, the depolarizing stimulus protocol was repeated at least 3 times, producing at least three traces of 120 s each. For analysis, each trace (excluding the depolarizing step) was divided into 0.5 s bins, computing the peak current within each bin, using the peak detection option in Signal 4 software, which subtracts from the maximum value within the bin, a mean baseline value for each bin. Within each trace, the peak current values measured from bins prior to the depolarizing step (baseline bins) were averaged and their mean was used to normalize the peak current values for all bins in a trace. For each neuron, the normalized peak current values for each bin (same time window) were averaged across traces, obtaining an average current value. Changes in IPSPs by DSI were assessed similarly, except that traces were divided into 0.1 s bins to minimize the effects of summation of overlapping IPSPs, which have slower decay kinetics than IPSCs, during detection by the peak measurement option in Signal 4 software. Suppression of IPSC or IPSP amplitude by DSI would directly decrease the peak current or peak potential values measured using this method. In fact, previous studies have shown that DSI strongly reduces the sIPSC amplitude at cannabinoid-sensitive synapses studied in isolation in synaptically connected pairs (Glickfeld & Scanziani, 2006; Neu et al. 2007; Galarreta et al. 2008). In addition, other studies have shown that DSI decreases IPSC frequency (Pitler & Alger, 1992b). In some of our experiments, DSI transiently decreased sIPSC frequency to zero (Fig. 2B). Our DSI measurement method also detects decreases in IPSC frequency, because the absence of sIPSCs within a bin is measured as a peak current amplitude equal to zero. In a group of experiments, we compared the magnitude of DSI determined using the method described above versus directly measuring sIPSC frequency. These results are shown in Supplemental Fig. 1 (available online only), demonstrating that measurements of DSI by these two methods produced highly correlated estimations of DSI magnitude. For classification of DSI-positive or DSI-negative cells we calculated the standard deviation for the baseline responses recorded before the depolarizing step by using the values of 1 s bins. A cell was considered to be DSI-positive when the two consecutive normalized IPSC values right after the depolarizing step (values at 1 and 2 s after the depolarization ended) satisfied the following criteria: (1) both normalized IPSC values were below 1, and (2) either IPSC value was less than 1 – 1 × SD of the baseline. To quantify the currents observed immediately following application of the depolarizing steps to induce DSI (Fig. 7), we divided each trace into 0.5 s bins and used the maximum value option in Signal 4 software to measure the holding current in each bin independently of the minimums represented by sIPSCs. For each trace, the initial holding current value was subtracted from the value measured in each bin. Depolarizing steps produce an outward current that is reduced by DAGLα deficiency A, the holding current for a holding potential of –80 mV was measured every 0.5 s starting 5 s before the depolarizing step (–80 to 0 mV, 4 s) used to induce DSI. The initial holding current value in each trace was subtracted from all values measured in the trace. The plot shows the changes in holding current relative to the initial value averaged across at least three traces per neuron and then across neurons (wild-type, n= 17 cells; DAGLα−/−, n= 14 cells; DAGLβ−/−, n= 13 cells). The current e" @default.
• W1645330008 created "2016-06-24" @default.
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• W1645330008 date "2011-10-14" @default.
• W1645330008 modified "2023-09-27" @default.
• W1645330008 title "Postsynaptic diacylglycerol lipase α mediates retrograde endocannabinoid suppression of inhibition in mouse prefrontal cortex" @default.
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